0steoporosis represents a major challenge to the maintenance of health and quality of life in an aging population. Among the most promising and least exploited therapeutic interventions are those treatments designed to increase bone density. The selection and validation of targets that can be manipulated to achieve this anabolic effect depends on an increasing amount of information relating to bone formation. A novel area for drug discovery - protein homeostatic regulation via ubiquitin pathway enzymes -- has recently been demonstrated to have relevance to the search for anabolic osteoporosis drugs. Prajal, a RING-finger E3 ubiquitin ligase, has been found to catalyse ubiquitination and, subsequently, induce proteasomal degradation of Dlxin-1, a coactivator of the transcriptional activator DIx5. DIx5 is associated with programmed bone formation. Thus, selective inhibitors of this E3 ligase are hypothesized to increase bone density. In Phase I, it is proposed to establish a yeast-based screening assay for inhibitors of Prajal. Essential components of the E3 system (Prajal) will be cloned and expressed in S. cerevisiae, along with human Dlxin-1 linked to p53, and a reporter construct that monitors p53 activity (beta-galactosidase activation). The reconstructed E3 ligase function and reporter system will then be configured and validated as a high throughput screen for inhibitors of Prajal-mediated Dlxin-1 ubiquitination. Collections of plant and marine organism extracts, along with a small compound collection, will be screened for potent inhibitors of Prajal E3 ubiquitin ligase activity. In Phase II, fractionation of active extracts will be guided by the assay, to identify active principles. Novel pure compounds arising from this effort will be considered as development candidates for osteoporosis. The modular assay construction format will permit evaluation of other E3s that are associated with a variety of diseases.